COMPOUNDS AND METHODS FOR INDUCING BROWNING OF WHITE ADIPOSE TISSUE

Description

Field of Invention

[0001] The present disclosure relates to compounds for inducing browning of white adipose tissue in vitro and in vivo, the preparation method thereof, as well as a composition comprising the same. Further, the present disclosure relates to the use of the compounds and the method to treat metabolic disorders, such as obesity and diabetes.

Background of the invention

[0002] Adipose tissues can be traditionally classified into white adipose tissue (WAT) and brown adipose tissue (BAT). While WAT stores nutrients as lipids, BAT can dissipate lipids to provide heat in a process called thermogenesis. BAT thermogenesis is dependent on the activation of uncoupling protein-1 (UCP1), which is located in the inner mitochondrial membrane of BAT. When activated, UCP1 uncouples oxidative phosphorylation in mitochondria to dissipate the electrochemical gradient as heat.

[0003] In addition to the "classical" and "developmentally programmed" brown adipocytes clustered in defined anatomical BAT depots, a "browning" process, which consists of the induced appearance of UCP1-expressing and multilocular brown adipocytes in WAT depots, often in response to specific stimuli such as prolonged cold exposure and chronic treatment with β3-adrenergic stimuli. These inducible brown adipocytes within classical WAT depots have been called "brite" (brown-in-white) or "beige" adipocytes. The important roles of brite/beige cells in thermoregulation and energy homeostasis are highlighted recently. In adult humans, the predominant form of brown adipocytes is brite/beige cells, which can be induced from WAT under proper stimulation. In light of the promising metabolic benefits of brown adipocytes, intense research has been conducted in recent years to search for physiological, pharmacological and dietary agents that can enhance browning of WAT by induction of UCP1 expression or/and mitochondrial oxidative metabolism. So far, dozens of "browning" agents have been reported, including the sympathetic activators (such as the β3-adrenergic agonists BRL26830A and CL-316243), prostaglandins, PPARα and PPARγ agonists, retinoids, activators of AMP-activated protein kinase, thyroid hormone, bone morphogenetic protein 7 (BMP7), irisin and fibroblast growth factor (FGF)21. While most of these agents exert dual effects on both activation of classical BAT and induction of WAT browning, and some of them (such as prostaglandin, irisin and FGF21) are specific to the browning of WAT. Although some drugs are found to induce browning, they may show strong side effect. Therefore, it is important to look for safer and more effective "browning" food supplements and/or drugs for healthy development and treatment of obesity as well as it associated metabolic disorders.

[0004] WO 2005/099673 discloses a genus of polycyclic thiazoles for use as modulators of potassium ion channels.

Summary of the invention

[0005] In one aspect, the present invention provides a compound or physiologically acceptable salt or hydrate or solvate thereof, wherein the compound is selected from the following compounds:

5. 3-(2-(pyridin-2-ylamino)thiazol-4-yl)benzonitrile;

13. N-(4-chloropyridin-2-yl)-4-(3,4-dimethoxyphenyl)thiazol-2-amine;

23. N-(pyridin-2-yl)-4-(4-(trifluoromethoxy)phenyl)thiazol-2-amine;

25. 4-(4-bromo-2-methoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

28. 4-(2-bromo-4-morpholinophenyl)-N-(pyridin-2-yl)thiazol-2-amine;

37. 1-isopropyl-3-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)urea;

38. N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)propionamide;

39. N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)butyramide;

42. 4-(4-isopropoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

43. 4-(4-butoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

44. ethyl 2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetate;

45. 2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetic acid;

46. 1-morpholino-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)ethanone;

47. N-ethyl-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetamide;

48. methyl 4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoate;

50. 4-(biphenyl-4-yl)-N-(pyridin-2-yl)thiazol-2-amine;

52. morpholino(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)methanone;

53. N-ethyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

54. 4-(4-(morpholinomethyl)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

55. 4-(4-(benzylamino)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

56. 4-(4-(neopentylamino)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

57. N-isopropyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

58. N-phenyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

59. (4-methylpiperazin-1-yl)(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)methanone;

60. 4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzyl acetate;

61. 1-(4-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)piperidin-1-yl)ethanone;

62. N-(2-(2-methoxyethoxy)ethyl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

64. 4-(2-(pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-3-yl)benzamide;

65. 4-(2-(pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-4-yl)benzamide;

66. N,N-diethyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

67. (S)-ethyl 1-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoyl)pyrrolidine-2-carboxylate;

69. N-(pyridin-2-yl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

72. 4-(4-(piperidin-1-ylmethyl)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

73. 1-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)ethanone;

74. N-hydroxy-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

76. N-(pyridin-2-yl)-4-(4-(thiophen-3-yl)phenyl)thiazol-2-amine;

77. 4-(4-(2H-tetrazol-5-yl)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

78. 4-(4-ethynylphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

79. N-(pyridin-2-yl)-4-(4-(pyridin-4-yl)phenyl)thiazol-2-amine;

83. 4-(4-amino-3-methylphenyl)-N-(pyridin-2-yl)thiazol-2-amine.

[0006] In another aspect, the present invention provides a pharmaceutical composition comprising the compound or physiologically acceptable salt or hydrate or solvate thereof of the present invention and at least one pharmaceutically acceptable carrier or diluent, optionally wherein said compound is in free form or in a pharmaceutically acceptable salt form. Such composition may be an oral composition, injectable composition or suppository. And the composition may be manufactured in a conventional manner by mixing, granulating or coating methods.

[0007] In an embodiment of the invention, the composition is an oral composition and it may be a tablet or gelatin capsule. Preferably, the oral composition comprises the present compound together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets, together with c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragamayth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; and if desired, d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) additives, e.g., absorbents, colorants, flavors and sweeteners.

[0008] In another embodiment of the invention, the composition is an injectable composition, and may be an aqueous isotonic solution or suspension.

[0009] In yet another embodiment of the invention, the composition is a suppository and may be prepared from fatty emulsion or suspension.

[0010] Preferably, the composition is sterilized and/or contains adjuvant. Such adjuvant can be preserving, stabilizing, wetting or emulsifying agent, solution promoter, salt for regulating the osmotic pressure, buffer and/or any combination thereof.

[0011] Alternatively or in addition, the composition may further contain other therapeutically valuable substances for different applications, like solubilizers, stabilizers, tonicity enhancing agents, buffers and/ or preservatives.

[0012] In an embodiment of the invention, the composition may be a formulation suitable for transdermal application. Such formulation includes an effective amount of the compound of the present invention and a carrier. Preferably, the carrier may include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. A transdermal device contain the formulation may also be used. The transdermal device may be in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Otherwise, a matrix transdermal formulation may also be used.

[0013] In another embodiment of the invention, the composition may be a formulation suitable for topical application, such as to the skin and eyes, and may be aqueous solution, ointment, cream or gel well known in the art.

[0014] The compound or composition according to the invention finds use in a method of inhibiting WAT secretion from a cell.

[0015] For therapeutical use, the compound of the present invention could be administered in a therapeutically effective amount via any acceptable way known in the art singly. As used herein, the therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. Generally, the satisfactory result is indicated to be obtained systemically at a daily dosage of about 0.03 to 2.5 mg/kg per body weight of the subject. In one embodiment, the indicated daily dosage for larger mammal as human is in the range from about 0.5mg to about 100mg. Preferably, the compound is administered in divided doses up to four times a day or in retard form. In another embodiment, suitable unit dosage forms for oral administration comprise from 1 to 100 mg active ingredient.

[0016] Alternatively, the compound of the present invention may be administered in a therapeutically effective amount as the active ingredient in combination with one or more therapeutic agents, such as pharmaceutical combinations. There may be synergistic effects when the compound of the present invention is used with a chemotherapeutic agent known in the art. The dosage of the co-administered compounds could vary depending on the type of co-drug employed, the specific drug employed, the condition being treated and so forth.

[0017] The compound of the present invention or the composition thereof may be administered by any conventional route. In one embodiment, it is administered enterally, such as orally, and in the form of tablets or capsules. In another embodiment, it is administered parenterally and in the form of injectable solutions or suspensions. In yet another embodiment, it is administered topically and in the form of lotions, gels, ointments or creams, or in a nasal or suppository form.

[0018] In another aspect, the disclosure also provides a pharmaceutical combination, preferably, a kit, comprising a) a first agent which is the compound of the present invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. In addition, the kit may comprise instructions for its administration.

[0019] The combination of the present disclosure may be used in vitro or in vivo. Preferably, the desired therapeutic benefit of the administration may be achieved by contacting cell, tissue or organism with a single composition or pharmacological formulation that includes the compound of the present invention and one or more agents, or by contacting the cell, tissue or organism with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another. The agents of the combination may be administered at the same time or separately within a period of time. Preferably, the separate administration can result in a desired therapeutic benefit. The present compound may precede, be co-current with and /or follow the other agents by intervals ranging from minutes to weeks. A person skilled in the art could generally ensure the interval of the time of each delivery, wherein the agents administered separately could still be able to exert an advantageously combined effect on the cell, tissue or organism. In one embodiment, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously as the candidate substance, i.e., with less than about one minute. In another embodiment, one or more agents may be administered about between 1 minute to 14 days.

[0020] In another aspect, the present disclosure provides a process for preparing the compound of the present invention or the salts or derivatives thereof.

[0021] The compound of the invention may be prepared following any one of the synthetic methodologies described below. In the reactions described, reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, may be protected to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice (see e.g., T.W. Greene and P. G. M. Wuts in "Protective Groups in Organic Chemistry", John Wiley and Sons, 1991). Suitable leaving groups for use in the synthetic methodologies described include halogen leaving groups and other conventional leaving groups known in the art. Preferably, the leaving group is chloro or bromo.

[0022] In another embodiment, the compound of the invention or the salts thereof may also be obtainable in the form of hydrates, or their crystals which may include for example the solvent used for crystallization (present as solvates). Salts can usually be converted to compounds in free form by treating with suitable basic agents, preferably with alkali metal carbonates, alkali metal hydrogen carbonates, or alkali metal hydroxides, more preferably with potassium carbonate or sodium hydroxide. A compound of the invention in a base addition salt form may be converted to the corresponding free acid by treating with a suitable acid, such as hydrochloric acid. In view of the close relationship between the novel compounds in free form and those in the form of their salts, including those salts that may be used as intermediates, for example in the purification or identification of the novel compounds, any reference to the free compounds is to be understood as referring also to the corresponding salts, as appropriate.

[0023] Salts of the present compound with a salt-forming group may be prepared in a manner known in the art. Acid addition salts of the compound may thus be obtained by treatment with an acid or with a suitable anion exchange reagent. Pharmaceutically acceptable salts of the compound of the invention may be formed as acid addition salts from the compound with a basic nitrogen atom with organic or inorganic acids.

[0024] Preferably, suitable inorganic acids include halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid.

[0025] Preferably, suitable organic acids include carboxylic, phosphoric, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid,-malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4 aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane-or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5-naphthalene-disuifonic acid, 2-, 3-or 4 methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N cyclohexylsulfamic acid, N-methyl-, N-ethyl-or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.

[0026] Alternatively, it is also possible to use pharmaceutically unacceptable salts for isolation or purification, for example picrates or perchlorates. But for therapeutic use, only pharmaceutically acceptable salts or free compounds are employed, where applicable in the form of pharmaceutical preparations.

[0027] The compound of the present invention in unoxidized form may be prepared from N-oxides of compound of the invention by treating with a reducing agent in a suitable inert organic solvent at 0 to 80°C. Preferably, the reducing agent is sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, or tribromide. Preferably, the invert organic solvent is acetonitrile, ethanol, or aqueous dioxane.

[0028] Protected derivatives of the compound of the present invention may be made by means known in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal may be found in T. W. Greene, "Protecting Groups in Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999.

[0029] The compounds of the present invention may be prepared as their individual stereoisomers. The process includes reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. Resolution of enantiomers may be carried out using covalent diastereomeric derivatives of the compound of the present invention, or by using dissociable complexes such as crystalline diastereomeric salts. Diastereomers have distinct physical properties presented by melting points, boiling points, solubilities, and reactivity, and may be readily separated by taking advantage of these dissimilarities. The diastereomers may be separated by fractionated crystallization, chromatography, or by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture may be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981.

[0030] In conclusion, the compound of the present invention could be made by the process described below; optionally a pharmaceutically acceptable salt may be converted from the compound of the present invention; optionally a pharmaceutically acceptable N-oxide may be converted from an unoxidized form of the compound the present invention; and optionally an individual isomer of the compound of the present invention is resolved from a mixture of isomers.Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the examples hereinafter. One of skill in the art will appreciate that the above transformations are only representative of methods for preparation of the compounds of the present invention, and that other well-known methods can similarly be used.

[0031] In another aspect, the present invention also provides a compound or physiologically acceptable salt or hydrate or solvate thereof as defined above or the composition thereof for use in treating metabolic disorders, such as obesity and diabetes.

Description of the Figures

[0032] Figure 1: Compound 1 promotes browning of adipose tissue from 16-week-old obese mice induced by high fat diets for 8-week. Hematoxylin and eosin staining (left panel) and UCP1 immunochemistry (right panel) analysis of adipose tissue in mice treated with compound 1 compared with control group, showing browning effects of white adipose tissue in the embodiment of reduced cell size and increased cell number.

Embodiments

[0033] 

Abbreviation

Definition or Explanation

DCE

1,2-Dichloroethane

DCM

Dichloromethane

DIPEA

N,N'-Diisopropylethylamine

DMF

N,N-Dimethylformamide

eq.

equivalents

EDCI

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlide

HOBt

N-hydroxybenzotriazole

NBS

N-bromosuccinimide

TEA

triethylamine

THF

tetrahydrofuran

Methods of Preparation

[0034] The compounds herein disclosed, including the compounds of the present invention, may be prepared by methods as those illustrated in the following scheme I to II. Starting materials are commercially available or prepared by the methods as those illustrated in the following scheme III to V. Additional methods for making selected compounds of the present invention are provided below. Solvents, temperatures, pressures and other reaction conditions may readily be selected by one of ordinary skill in the art.

wherein:

each of Ring A and Ring B are independently absent; or independently selected from the group consisting of phenyl, 3-7 membered saturated or partially unsaturated carbocyclic ring, 8-10 membered bicyclic saturated or partially unsaturated carbocyclic ring, 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from N, O and S, 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from N, O, and S, 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from N, O and S, or 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from N, O and S;

each of R₁ and R₂ are independently selected from halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, -OR₃, -SR₃, -N(R₄)₂, -CN, -NO₂, -C(O)R₃, -C(S)R₃, -C(O)N(R₄)₂, -C(O)SR₃, -C(O)C(O)R₃, -C(O)CH₂C(O)R₃, -C(S)N(R₄)₂, -C(S)OR₃, -S(O)R₃, -SO₂N(R₄)₂, -N(R₄)C(O)R₃, -N(R₄)C(O)N(R₄)₂, -N(R₄)C(S)N(R₄)₂, -N(R₄)SO₂R₃, -N(R₄)SO₂N(R₄)₂, -N(R₄)N(R₄)₂, -N(R₄)C(=N(R₄))N(R₄)₂, -C=NN(R₄)₂, -C=NOR₃, -C(=N(R₄))N(R₄)₂, -OC(O)R₃, or -OC(O)N(R₄)₂;

each of m and n are independently 0-4, as valency permits, and

each of R₃ and R₄ are independently hydrogen or an optionally substituted group selected from C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl, each of which may be optionally substituted with halogen, amino, hydroxyl, alkoxy or cyano.

General protocol for the thiazole compound 3 formation

[0035] To a solution of substituted thiourea 1 (1.0 eq.) in alcoholic solvent (about 0.5 M concentration) such as methanol, ethanol or isopropanol was added substituted α-bromoketone 2 (1.0 eq.), and the mixture was heated to reflux for 3~8h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give compound 3 in 60~95% yield, such as the compounds 1-28, 31 and 33-83 listed below.

Step 1 General protocol for the 2-aminothiazole 5 formation

[0036] To a solution of thiourea 4 (1.0 eq.) in alcoholic solvent (about 0.5 M concentration) such as methanol, ethanol or isopropanol was added substituted α-bromoketone 2 (1.0 eq.), and the mixture was heated to reflux for 3~8h until the starting material was consumed. Then the mixture was cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 2-aminothiazole 5 of scheme II in 60~95% yield.

Step 2 General protocol for the thiazole compound 6 formation

[0037] 2-aminothiazole 5 (1.0 eq.) obtained from step 1 of scheme II was reacted with substituted acyl chloride (1.5 eq.) in the presence of a base (2.2 eq.) such as TEA, DIPEA, Na₂CO₃ in organic solvent (about 0.5 M concentration) such as DCM, DMF or CH₃CN to form amide compound 6 in 60~95% yield, such as the compounds 29 and 30 listed below.

[0038] or 2-aminothiazole 5 (1.0 eq.) obtained from step 1 of scheme II was coupled with substituted carboxylic acid (1.1 eq.) in the presence of classical coupling reagents (1.1 eq.) in amide synthesis such as EDCI/HOBt/DIPEA combination in organic solvents (about 0.5 M concentration) such as DCM, THF or DMF to afford amide compound 6 in 55~85% yield, such as the compounds 32 listed below.

General procedure for the preparation of thiourea intermediates 1 from amines

[0039] The substituted thiourea intermediates 1 of scheme I were commercially available or prepared according to procedures provided in the following scheme III.

[0040] Phenyl isothiocyanate (1.1 eq.) was added dropwise to a stirred solution of amine 7 (1.0 eq.) in organic solvent (about 0.1 M concentration) such as acetone, THF or CH₃CN at room temperature. The reaction mixture was heated to reflux for 1-3 h until the amine 7 was consumed. Then the mixture was cooled to room temperature, poured into water-ice, and stirred for an additional 15 min. The benzoyl thiourea precipitate was collected by filtration and washed with more water. This crude material was dissolved in methanol (about 0.05M concentration) and treated with aqueous 1N NaOH (2 eq.) and heated to 80°C until the hydrolysis was completed. After cooling, the mixture was poured into water-ice and sufficient aqueous IN HC1 was added to produce a neutral (pH about 7) solution. The thiourea intermediate typically precipitates from the neutral solution and was collected by filtration and dried. This two-step procedure provides thiourea intermediates in 50~95% overall yield. The thiourea intermediates can be used directly in the next step without further purification.

General procedure for the preparation of substituted α-bromoketone intermediates 2

[0041] The substituted α-bromoketone intermediates 2 of schemes I to II were commercially available or prepared according to procedures provided in the following scheme IV to V.

[0042] To a solution of ketone 8 (1.0 eq.) in organic solvent (about 0.5M concentration) such as DCM, chloroform or ether was added bromine (1.0 eq.) dropwise at 0°C. The mixture was then warmed to room temperature or elevated temperature (50~90 °C) if necessary and stirred for 2~6h until the ketone was consumed. The mixture was then extracted, dried, evaporated and purified by silica gel chromatography to give compound 2 of scheme IV in 70~95% yield.

[0043] When ketones were commercially unavailable, α-bromoketoneintermediates 2 were prepared in an alternative way provided in the following scheme V.

Step 1 General procedure for the preparation of ethoxyethene intermediates 10 via stile coupling reaction

[0044] To a solution of halogenated compound 9 (1.0 eq.) in organic solvent (about 1M concentration) such as dioxane, DCE or toluene was added tributyl (1-ethoxyvinyl)tin (1.5 eq.) and dichlorobis(triphenylphosphine)palladium (II) (0.05 eq.). The mixture was purged with nitrogen for 5min, and stirred at elevated temperature (90~120°C) in a sealed tube for 12~18h until the compound 9 was consumed. The mixture was then cooled to room temperature, extracted, dried, evaporated and purified by silica gel chromatography to give compound 10 of scheme V in 65~85% yield.

Step 2 General procedure for the preparation of α-bromoketoneintermediates 2 via bromination of intermediates 10

[0045] To a solution of compound 10 (1.0 eq.) in THF/H₂O (about 0.5M concentration, V_THF:V_H2O=3:1) was added NBS (1.0 eq.). The mixture was stirred at room temperature for 10~30min, then extracted, dried, evaporated and purified by silica gel chromatography to afford α-bromoketone intermediates 2 of scheme V in 55~75% yield.

Detailed embodiments

[0046] The present invention is further exemplified by the following and examples that illustrate the preparation of the compounds of the invention.

[0047] The invention will now be further described by the following working examples, which are preferred embodiments of the invention. All temperatures are in degrees Celsius (°C) unless otherwise indicated. Preparative Reverse Phase (RP) HPLC purifications were done on C18 reverse phase (RP) columns using water/methanol mixtures. All the synthesized compounds were characterized by at least NMR or LC/MS. During work up of reactions, the organic extract was dried over sodium sulfate, purified by silica gel column chromatography or (RP) HPLC, unless mentioned otherwise.

[0048] These examples are illustrative rather than limiting and it is to be understood that there may be other embodiments that fall within the scope of the claims appended hereto.

[0049] Compounds 1-4, 6-12, 14-22, 24, 26, 27, 29-36, 40, 41, 49, 51, 63, 68, 70, 71, 75 and 80-82 do not form part of the claimed invention.

Example 1

The preparation of compound 1

[0050] 

Step 1 Preparation of intermediate 1-1

[0051] Phenyl isothiocyanate (13.52g, 0.1mol) was added dropwise to a stirred solution of 2-aminopyridine (9.41g, 0.1mol) in 200mL acetone at room temperature. The reaction mixture was heated to reflux for 3 h until the 2-aminopyridine was consumed. Then the mixture was cooled to room temperature, poured into water-ice, and stirred for an additional 15 min. The benzoyl thiourea precipitate was collected by filtration and washed with more water. This crude material was dissolved in 200mL methanol and treated with 200mL aqueous IN NaOH and heated to 80°C until the hydrolysis was completed. After cooling, the mixture was poured into water-ice and sufficient aqueous IN HClwas added to produce a neutral (pH about 7) solution. The thiourea intermediate precipitate was collected by filtration and dried, providing 13.9g of intermediate 1-1 in 91% yield. ESI-MS m/z 154.0 [M+H].

Step 2 Preparation of Compound 1

[0052] To a solution of intermediate 1-1 (0.153g, 1.0mmol) in 5mL ethanol was added 2-bromo-1-(3,4-dimethoxyphenyl)ethanone 2 (0.259g, 1.0mmol), and the mixture was heated to reflux for 3h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 0.266g of compound 1 in 85% yield. ¹H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 8.34 (dd, J = 5.0, 1.0 Hz, 1H), 7.49-7.39 (m, 3H), 6.94 (s, 1H), 6.90-6.86 (m, 1H), 6.86-6.80 (m, 1H), 6.58 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H). ESI-MS m/z 314.3 [M+H].

Example 2

The preparation of compound 18

[0053] 

[0054] To a solution of intermediate 1-1 (0.153g, 1.0mmol) in 5mL ethanol was added 2-bromo-1-(3,5-dimethoxyphenyl)ethanone intermediate 18-1 (0.259g, 1.0mmol), and the mixture was heated to reflux for 3h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 0.261g of compound 18 in 84% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.88 (s, 1H), 8.46-8.09 (m, 1H), 7.47-7.32 (m, 3H), 7.16 (s, 1H), 6.98 (d, J= 2.4 Hz, 2H), 6.49 (d, J= 2.4 Hz, 1H), 3.94 (s, 3H), 3.82 (s, 3H). ESI-MS m/z 314.3 [M+H].

Example 3

The preparation of compound 32

[0055] 

Step 1 Preparation of intermediate 32-1

[0056] To a solution of thiourea (7.61g, 0.1mol) in 200mL ethanol was added intermediate 1-1 (25.9g, 0.1mol), and the mixture was heated to reflux for 3~8h until the starting material was consumed. Then the mixture was cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 18.9g of intermediate 32-1 in 85% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.38-7.27 (m, 2H), 6.88 (d, J= 8.2 Hz, 1H), 6.60 (s, 1H), 5.04 (s, 2H), 3.95 (s, 3H), 3.90 (s, 3H). ESI-MS m/z 237.3 [M+H].

Step 2 Preparation of compound 32

[0057] To a solution of 2-picolinic acid (21.17mg, 0.172mmol) in 5mL DCM were added EDCI (36.1mg, 0.189mmol), HOBt (25.5mg, 0.189mmol), DIPEA (48.9mg, 0.378mmol) and the mixture was stirred at room temperature for 15min. Then intermediate 32-1 (40.6mg, 0.172mmol) was added. The mixture was continued to stir at room temperature for 12h until intermediate 32-1 was consumed, then evaporated, under reduced pressure and the crude material was purified by silica gel chromatography to give 45.8mg of compound 32 in 78% yield. ¹H NMR (400 MHz, CDCl₃) δ 11.23 (s, 1H), 8.69 (d, J = 4.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.96 (t, J = 7.0 Hz, 1H), 7.55 (dd, J = 7.4, 5.0 Hz, 1H), 7.47 (s, 1H), 7.46-7.41 (m, 1H), 7.11 (s, 1H), 6.93 (d, J= 8.3 Hz, 1H), 3.99 (s, 3H), 3.93 (s, 3H). ESI-MS m/z 342.3 [M+H].

Example 4

The preparation of compound 36

[0058] 

Step 1 Preparation of compound 34

[0059] To a solution of intermediate 1-1 (15.3g, 0.1mol) in 200mL ethanol was added intermediate 36-1 (24.4g, 0.1mol), and the mixture was heated to reflux for 3h until the starting material was consumed. The mixture was then cooled to room temperature, evaporated under reduced pressure and purified by silica gel chromatography to give 27.98g of compound 34 in 94% yield. ¹H NMR (400 MHz, DMSO) δ 11.53 (s, 1H), 8.30 (dd, 12.9, 6.7 Hz, 3H), 8.16 (d, 8.7 Hz, 2H), 7.80 (s, 1H), 7.73 (t, J = 7.1 Hz, 1H), 7.11 (d, J= 8.3 Hz, 1H), 7.02-6.86 (m, 1H). ESI-MS m/z 299.3 [M+H].

Step 2 Preparation of compound 35

[0060] To a solution of compound 34 (27.98g, 93.8mmol) in 200mL acetic acid was added iron powder (15.5g, 281mmol) in portions. Upon complete addition, the mixture was carefully heated to 100°C. After 3 h, the mixture was cooled to room temperature and filtered through Celite (TM). The Celite (TM) was washed with EtOH and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to give compound 35 in 88% yield. ¹H NMR (400 MHz, DMSO) δ 11.25 (s, 1H), 8.29 (d, J= 4.0 Hz, 1H), 7.77-7.60 (m, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.94-6.79 (m, 1H), 6.58 (d, J = 8.5 Hz, 2H), 5.18 (s, 2H). ESI-MS m/z 267.3 [M+H].

Step 3 Preparation of compound 36

[0061] To a solution of compound 35 (133.2mg, 0.5mmol) in 5mL pyridine was added acetyl chloride (58.9mg, 0.75mmol) at 0°C. After addition, the mixture was warmed to room temperature and stirred for 8h until compound 35 was consumed. The mixture was then extracted, dried, evaporated and purified by column chromatography to give 132mg of compound 36 in 85% yield. ¹H NMR (400 MHz, DMSO) δ 11.37 (s, 1H), 9.99 (s, 1H), 8.30 (d, J= 4.9 Hz, 1H), 7.82 (d, J= 8.6 Hz, 2H), 7.70 (dd, J= 11.2, 4.3 Hz, 1H), 7.62 (d, 8.6 Hz, 2H), 7.29 (s, 1H), 7.09 (d, 8.3 Hz, 1H), 7.00-6.84 (m, 1H), 2.06 (s, 3H). ESI-MS m/z 311.3 [M+H].

[0062] The following compounds were prepared using a procedure similar to that described above.

No.	Structure	NMR/MS
1		1H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 8.34 (dd, J = 5.0, 1.0 Hz, 1H), 7.49-7.39 (m, 3H), 6.94 (s, 1H), 6.90-6.86 (m, 1H), 6.86-6.80 (m, 1H), 6.58 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H). ESI-MS m/z 314.3 [M+H].
2		1H NMR (400M, DMSO) δ 11.42 (br, 1H), 8.21 (d, J= 5.49 Hz, 1H), 7.49 (s, 1H), 7.46-7.39 (m, 1H), 7.31 (s, 1H), 6.98 (d, J= 8.2 Hz, 1H), 6.93 (s, 1H), 6.83 (d, J= 4.9 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 3H), 2.39 (s, 3H). ESI-MS m/z 328.3 [M+H].
3		1H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 8.39-8.31 (m, 1H), 7.49-7.39 (m, 3H), 6.94 (s, 1H), 6.90-6.86 (m, 1H), 6.86-6.80 (m, 2H), 6.58 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H). ESI-MS m/z 284.3 [M+H].
4		1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 8.27 (d, J=5.0 Hz, 1H), 7.86-7.79 (m, 2H), 7.41-7.29 (m, 4H), 6.98 (s, 1H), 6.85-6.79 (m, 1H), 6.51 (d, J= 8.0Hz, 1 H ). ESI-MS m/z 254.3 [M+H].
5		1H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.38-8.30 (m, 1H), 7.49-7.39 (m, 3H), 6.98 (s, 1H), 6.90-6.86 (m, 1H), 6.83-6.79 (m, 2H), 6.59 (d, J = 8.3 Hz, 1H). ESI-MS m/z 279.3 [M+H].
6		ESI-MS m/z 365.3 [M+H].
7		ESI-MS m/z 293.3 [M+H].
8		ESI-MS m/z 284.3 [M+H].
9		ESI-MS m/z 255.3 [M+H].
10		1H NMR (400 MHz, CDCl3) δ 10.55 (s, 1H), 7.66-7.59 (m, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.03 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.89-6.81 (m, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 2.41 (s, 3H). ESI-MS m/z 328.3 [M+H].
11		1H NMR (400M, DMSO) δ 11.39 (br, 1H), 8.18 (s, 1H), 7.61 (d, J= 8.2 Hz, 1H), 7.49-7.40 (m, 2H), 7.34 (s, 1H), 7.01 (d, J= 8.2 Hz, 1H), 6.98 (d, J= 8.2 Hz, 1H), 3.91 (s, 3H), 3.88 (s, 3H), 2.34 (s, 3H). ESI-MS m/z 328.3 [M+H].
12		ESI-MS m/z 328.3 [M+H].
13		ESI-MS m/z 348.9 [M+H].
14		ESI-MS m/z 384.3 [M+H].
15		ESI-MS m/z 314.3 [M+H].
16		ESI-MS m/z 297.3 [M+H].
17		ESI-MS m/z 344.3 [M+H].
18		1H NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 8.46-8.09 (m, 1H), 7.47-7.32 (m, 3H), 7.16 (s, 1H), 6.98 (d, J = 2.4 Hz, 2H), 6.49 (d, J = 2.4 Hz, 1H), 3.94 (s, 3H), 3.82 (s, 3H). ESI-MS m/z 314.3 [M+H].
19		ESI-MS m/z 328.3 [M+H].
20		1H NMR (400 MHz, CDCl3) δ 7.52 (s, 1H), 7.41 (d, J = 6.9 Hz, 2H), 7.36 (dd, J = 8.6, 5.7 Hz, 4H), 7.13-7.04 (m, 1H), 6.90 (d, J = 8.9 Hz, 1H), 6.71 (s, 1H), 3.95 (s, 3H), 3.92 (s, 3H). ESI-MS m/z 313.3 [M+H].
21		1H NMR (400 MHz, CDCl3) δ 10.11 (s, 1H), 8.33 (d, J = 4.5 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 6.1 Hz, 1H), 7.19 (d, J = 7.8 Hz, 2H), 6.99 (s, 1H), 6.82 (d, J= 5.1 Hz, 1H), 6.54 (s, 1H), 2.39 (s, 3H). ESI-MS m/z 268.3 [M+H].
22		1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 8.34 (d, J = 4.6 Hz, 1H), 7.85 (dd, J = 8.7, 5.5 Hz, 2H), 7.44 (t, J = 7.7 Hz, 1H), 7.07 (t, J= 8.6 Hz, 2H), 6.97 (s, 1H), 6.89-6.79 (m, 1H), 6.58 (d, J = 5.7 Hz, 1H). ESI-MS m/z 272.3 [M+H].
23		1H NMR (400 MHz, CDCl3) δ 10.19 (s, 1H), 8.33 (t, J = 7.5 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.38 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.04 (s, 1H), 6.87-6.76 (m, 1H), 6.47 (d, J = 8.3 Hz, 1H). ESI-MS m/z 338.3 [M+H].
24		1H NMR (400 MHz, CDCl3) δ 9.33 (s, 1H), 8.35 (d, J = 4.5 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.54-7.39 (m, 2H), 7.27 (d, J = 11.3 Hz, 1H), 7.01 (t, J = 8.0 Hz, 2H), 6.90-6.79 (m, 1H), 6.73 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H). ESI-MS m/z 283.3 [M+H].
25		1H NMR (400 MHz, CDCl3) δ 9.36 (s, 1H), 8.35 (d, J= 4.5 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.54-7.39 (m, 2H), 7.27 (d, J = 11.3 Hz, 1H), 7.01 (t, J = 8.0 Hz, 2H), 6.90-6.79 (m, 1H), 6.73 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H). ESI-MS m/z 363.3 [M+H].
26		1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.63 (d, J = 5.1 Hz, 2H), 8.37 (d, J = 3.9 Hz, 1H), 7.75 (d, J= 5.0 Hz, 2H), 7.56 (t, J = 7.2 Hz, 1H), 7.29 (s, 1H), 6.99-6.81 (m, 1H), 6.74 (d, J = 8.2 Hz, 1H). ESI-MS m/z 255.3 [M+H].
27		1H NMR (400 MHz, CDCl3) δ 9.44 (s, 1H), 8.63 (d, J= 4.5 Hz, 1H), 8.36 (d, J = 4.8 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.61 (s, 1H), 7.51 (dd, J = 11.1, 4.4 Hz, 1H), 7.22-7.14 (m, 1H), 6.93-6.83 (m, 1H), 6.72 (d, J = 8.3 Hz, 1H). ESI-MS m/z 255.3 [M+H].
28		1H NMR (400 MHz, DMSO) δ 11.40 (s, 1H), 8.30 (d, 4.6 Hz, 1H), 8.16 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.71 (t, J= 7.4 Hz, 1H), 7.43 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 7.04-6.78 (m, 1H), 3.76 (s, 4H), 3.00 (s, 4H). ESI-MS m/z 418.3 [M+H].
29		1H NMR (400 MHz, CDCl3) δ 10.55 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.03 (s, 1H), 6.91 (d, J = 8.3 Hz, 1H), 3.98 (s, 3H), 3.87 (s, 3H), 2.00 (s, 3H). ESI-MS m/z 279.3 [M+H].
30		1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.08 (s, 1H), 6.91 (d, J = 8.0 Hz, 1H), 3.96 (s, 3H), 3.92 (s, 3H). ESI-MS m/z 341.3 [M+H].
31		1H NMR (400 MHz, CDCl3) δ 7.38 (s, 1H), 7.34 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.00 (s, 1H), 3.90 (d, J = 4.9 Hz, 6H), 2.88 (s, 3H). ESI-MS m/z 251.3 [M+H].
32		1H NMR (400 MHz, CDCl3) δ 11.23 (s, 1H), 8.69 (d, J = 4.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.96 (t, J = 7.0 Hz, 1H), 7.55 (dd, J = 7.4, 5.0 Hz, 1H), 7.47 (s, 1H), 7.46-7.41 (m, 1H), 7.11 (s, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.93 (s, 3H). ESI-MS m/z 342.3 [M+H].
33		1H NMR (400 MHz, DMSO) δ 10.45 (s, 1H), 8.84 (d, J = 2.5 Hz, 1H), 8.27 (d, J = 9.4 Hz, 1H), 8.17 (d, J = 3.4 Hz, 1H), 7.59-7.45 (m, 2H), 7.38 (dd, J = 8.3, 4.6 Hz, 1H), 7.29 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H). ESI-MS m/z 314.3 [M+H].
34		1H NMR (400 MHz, DMSO) δ 11.53 (s, 1H), 8.30 (dd, J = 12.9, 6.7 Hz, 3H), 8.16 (d, J = 8.7 Hz, 2H), 7.80 (s, 1H), 7.73 (t, J = 7.1 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.02-6.86 (m, 1H). ESI-MS m/z 299.3 [M+H].
35		1H NMR (400 MHz, DMSO) δ 11.25 (s, 1H), 8.29 (d, 4.0 Hz, 1H), 7.77-7.60 (m, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.94-6.79 (m, 1H), 6.58 (d, J = 8.5 Hz, 2H), 5.18 (s, 2H). ESI-MS m/z 267.3 [M+H].
36		1H NMR (400 MHz, DMSO) δ 11.37 (s, 1H), 9.99 (s, 1H), 8.30 (d, J = 4.9 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.70 (dd, J = 11.2, 4.3 Hz, 1H), 7.62 (d, J= 8.6 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 7.00-6.84 (m, 1H), 2.06 (s, 3H). ESI-MS m/z 311.3 [M+H].
37		1H NMR (400 MHz, DMSO) δ 11.35 (s, 1H), 8.35 (s, 1H), 8.30 (d, J = 4.7 Hz, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.22 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.99-6.84 (m, 1H), 6.02 (d, J = 7.5 Hz, 1H), 3.76 (dd, J = 13.4, 6.7 Hz, 1H), 1.10 (d, J = 6.5 Hz, 6H). ESI-MS m/z 354.3 [M+H].
38		1H NMR (400 MHz, DMSO) δ 11.37 (s, 1H), 9.92 (s, 1H), 8.30 (d, J = 4.0 Hz, 1H), 7.79 (t, J = 21.9 Hz, 2H), 7.77-7.67 (m, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.98-6.85 (m, 1H), 2.33 (q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H). ESI-MS m/z 325.3 [M+H].
39		1H NMR (400 MHz, DMSO) δ 11.37 (s, 1H), 9.92 (s, 1H), 8.44-8.21 (m, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.75-7.67 (m, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.29 (s, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.92 (dd, J = 6.8, 5.5 Hz, 1H), 2.30 (t, J = 7.3 Hz, 2H), 1.62 (dd, J = 14.7, 7.4 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). ESI-MS m/z 339.3 [M+H].
40		1H NMR (400 MHz, DMSO) δ 11.42 (s, 1H), 8.31 (dd, J = 5.1, 1.1 Hz, 1H), 7.89-7.84 (m, 2H), 7.75-7.69 (m, 1H), 7.64-7.59 (m, 2H), 7.50 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.96-6.91 (m, 1H). ESI-MS m/z 333.1 [M+H].
41		1H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H), 8.35 (d, J = 4.2 Hz, 1H), 7.81 (t, J = 7.3 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.22 (s, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.01(t, J = 6.4 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H). ESI-MS m/z 270.3 [M+H].
42		1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 8.30 (dd, J = 5.0, 1.1 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.8, 7.3, 1.9 Hz, 1H), 7.23 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.92-6.89 (m, 1H), 4.64 (dt, J = 12.0, 6.0 Hz, 1H), 1.28 (d, J = 6.0 Hz, 6H). ESI-MS m/z 312.3 [M+H].
43		1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 8.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.8, 7.2, 1.9 Hz, 1H), 7.24 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 0.8 Hz, 1H), 4.00 (t, J = 6.5 Hz, 2H), 1.76-1.65 (m, 2H), 1.51-1.38 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). ESI-MS m/z 326.3 [M+H].
44		1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 8.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.8, 7.2, 1.9 Hz, 1H), 7.27 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.9 Hz, 2H), 6.92 (dd, J = 6.7, 5.5 Hz, 1H), 4.81 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). ESI-MS m/z 356.3 [M+H].
45		1H NMR (400 MHz, DMSO) δ 13.01 (s, 1H), 11.37 (s, 1H), 8.30 (d, J = 4.0 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.76-7.67 (m, 1H), 7.26 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.92 (dd, J = 6.9, 5.7 Hz, 1H), 4.71 (s, 2H). ESI-MS m/z 328.3 [M+H].
46		1H NMR (400 MHz, DMSO) δ 11.35 (s, 1H), 8.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.70 (ddd, J = 8.9, 5.4, 1.9 Hz, 1H), 7.26 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.00-6.94 (m, 2H), 6.95-6.89 (m, 1H), 4.86 (s, 2H), 3.66-3.53 (m, 4H), 3.47 (s, 4H). ESI-MS m/z 397.4 [M+H].
47		1H NMR (400 MHz, DMSO) δ 9.49 (s, 1H), 8.41 (d, J = 3.7 Hz, 1H), 8.26 (t, J = 5.5 Hz, 1H), 7.82 (dt, J = 8.9, 5.4 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.22 (s, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.05 (dd, J = 7.0, 5.0 Hz, 1H), 6.78 (d, J = 8.6 Hz, 2H), 5.02 (s, 2H), 3.20-3.07 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H). ESI-MS m/z 355.3 [M+H].
48		1H NMR (400 MHz, DMSO) δ 11.50 (s, 1H), 8.32 (d, J = 4.4 Hz, 1H), 8.04 (q, J = 8.6 Hz, 4H), 7.74 (t, J = 7.0 Hz, 1H), 7.66 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.98-6.93 (m, 1H), 3.87 (s, 3H). ESI-MS m/z 312.3 [M+H].
49		1H NMR (400 MHz, DMSO) δ 11.51 (s, 1H), 8.32 (dd, J = 5.1, 1.1 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.93-7.85 (m, 2H), 7.79-7.71 (m,2H), 7.12 (d, J = 8.3 Hz, 1H), 6.96 (dd, J = 6.7, 5.5 Hz, 1H). ESI-MS m/z 279.3 [M+H].
50		1H NMR (400 MHz, DMSO) δ 11.59 (s, 1H), 8.35 (d, J = 4.3 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.82-7.69 (m, 5H), 7.53 (s, 1H), 7.48 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.03-6.96 (m, 1H). ESI-MS m/z 330.3 [M+H].
51		1H NMR (400 MHz, DMSO) δ 11.57 (s, 1H), 8.33 (dd, J = 5.1, 1.1 Hz, 1H), 8.22-8.14 (m, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.79-7.71 (m, 2H), 7.12 (d, J = 8.4 Hz, 1H), 6.97 (dd, J = 6.5, 5.2 Hz, 1H), 3.24 (s, 3H). ESI-MS m/z 332.4 [M+H].
52		1H NMR (400 MHz, DMSO) δ 11.43 (s, 1H), 8.32 (d, 4.0 Hz, 1H), 7.97 (t, J = 6.3 Hz, 2H), 7.77-7.68 (m, 1H), 7.54 (s, 1H), 7.47 (d, J = 8.3 Hz, 2H), 7.11 (d, J= 8.3 Hz, 1H), 6.95 (dd, J = 6.7, 5.5 Hz, 1H), 3.61-3.49 (m, 8H). ESI-MS m/z 367.3 [M+H].
53		1H NMR (400 MHz, DMSO) δ 11.44 (s, 1H), 8.47 (t, J = 5.4 Hz, 1H), 8.32 (d, J = 4.0 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.76-7.69 (m, 1H), 7.58 (s, 1H), 7.11 (d,J= 8.3 Hz, 1H), 6.95 (dd, J = 6.7, 5.5 Hz, 1H), 1.14 (t, J= 7.2 Hz, 3H). ESI-MS m/z 325.3 [M+H].
54		1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H), 8.34 (d, J = 4.9 Hz, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.58-7.50 (m, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.01 (s, 1H), 6.90- 6.83 (m, 1H), 6.77 (d, J = 8.3 Hz, 1H), 3.78-3.69 (m, 4H), 3.57 (s, 2H), 2.51 (s, 4H). ESI-MS m/z 353.4 [M+H].
55		1H NMR (400 MHz, DMSO) δ 11.25 (s, 1H), 8.28 (d, J = 4.0 Hz, 1H), 7.72-7.63 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.41-7.33 (m, 4H), 7.29-7.17 (m, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 6.90 (dd, J = 6.6, 5.6 Hz, 1H), 6.61 (d, J = 8.7 Hz, 2H), 6.38 (t, J = 6.0 Hz, 1H), 4.31 (d, J = 6.0 Hz, 2H). ESI-MS m/z 359.3 [M+H].
56		1H NMR (400 MHz, DMSO) δ 11.26 (s, 1H), 8.29 (d, J = 4.1 Hz, 1H), 7.74-7.63 (m, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.94-6.84 (m, 1H), 6.66 (d, J = 8.7 Hz, 2H), 5.58 (t, J = 6.0 Hz, 1H), 2.85 (d, J = 6.0 Hz, 2H), 0.96 (s, 9H).ESI-MS m/z 339.3 [M+H].
57		1H NMR (400 MHz, DMSO) δ 11.44 (s, 1H), 8.31 (d, J = 4.0 Hz, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.78-7.64 (m, 1H), 7.57 (s, 1H), 7.10 (d,J= 8.3 Hz, 1H), 7.00-6.82 (m, 1H), 4.11 (dd, J = 14.0, 6.7 Hz, 1H), 1.18 (d, J = 6.6 Hz, 6H). ESI-MS m/z 339.3 [M+H].
58		1H NMR (400 MHz, DMSO) δ 11.47 (s, 1H), 10.25 (s, 1H), 8.32 (d, J = 4.1 Hz, 1H), 8.09-8.02 (m, 4H), 7.80 (d, J = 8.3 Hz, 2H), 7.73 (t, J = 7.7 Hz, 1H), 7.64 (s, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.23-7.03 (m, 2H), 6.99-6.83 (m, 1H). ESI-MS m/z 373.3 [M+H].
59		1H NMR (400 MHz, DMSO) δ 11.43 (s, 1H), 8.50-8.13 (m, 1H), 7.97 (d, J = 8.2 Hz, 2H), 7.81-7.64 (m, 1H), 7.51 (s, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 6.6, 5.4 Hz, 1H), 3.66-3.57 (br, 4H), 2.44-2.35 (br, 4H), 2.19 (s, 3H). ESI-MS m/z 380.3 [M+H].
60		ESI-MS m/z 326.3 [M+H].
61		1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 8.30 (dd, J = 4.8, 1.0 Hz, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.75-7.66 (m, 1H), 7.35 (s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.4 Hz, 1H), 6.92 (dd, J = 6.7, 5.5 Hz, 1H), 4.59-4.49 (m, 1H), 3.92 (d, J = 13.4 Hz, 1H), 3.13 (t, J = 11.9 Hz, 1H), 2.78 (t, J = 12.0 Hz, 1H), 2.63-2.59 (m, 1H), 2.03 (s, 3H), 1.91-1.82 (m, 2H), 1.69-1.60 (m, 1H), 1.49-1.42 (m, 1H). ESI-MS m/z 379.3 [M+H].
62		1H NMR (400 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.51 (t, J = 5.5 Hz, 1H), 8.31 (d, J = 4.0 Hz, 1H), 7.99 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.76-7.68 (m, 1H), 7.58 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.97-6.90 (m, 1H), 3.59-3.50 (m, 4H), 3.48-3.39 (m, 4H), 3.24 (s, 3H). ESI-MS m/z 399.4 [M+H].
63		1H NMR (400 MHz, DMSO) δ 11.53 (s, 1H), 8.33 (d, J = 4.0 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.82-7.72 (m, 1H), 7.56-7.42 (m, 3H), 7.14 (d, J = 8.4 Hz, 1H), 7.04-6.89 (m, 1H). ESI-MS m/z 288.3 [M+H].
64		1H NMR (400 MHz, CDCl3) δ 10.56 (s, 1H), 9.87 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.46-8.39 (m, 2H), 7.85 (t, J= 7.9 Hz, 1H), 7.69-7.58 (m, 5H), 7.20-7.14 (m, 1H), 7.05 (s, 1H), 6.95-6.91 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H). ESI-MS m/z 374.4 [M+H].
65		1H NMR (400 MHz, DMSO) δ 11.47 (s, 1H), 10.59 (s, 1H), 8.49 (d, J = 6.2 Hz, 2H), 8.32 (d, J = 4.1 Hz, 1H), 8.07 (q, J = 8.5 Hz, 4H), 7.81 (d, J = 6.3 Hz, 2H), 7.72 (dd, J = 11.3, 4.2 Hz, 1H), 7.66 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.98-6.92 (m, 1H). ESI-MS m/z 374.4 [M+H].
66		ESI-MS m/z 353.4 [M+H].
67		ESI-MS m/z 443.4 [M+H].
68		1H NMR (400 MHz, DMSO) δ 11.49 (s, 1H), 9.91 (s, 1H), 8.62-8.36 (m, 1H), 7.86-7.77 (m, 3H), 7.77-7.69 (m, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.3 Hz, 1H), 6.97 (dd, J = 6.7, 5.4 Hz, 1H). ESI-MS m/z 408.0 [M+H].
69		1H NMR (400 MHz, CDCl3) δ 10.56 (s, 1H), 9.87 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.46-8.39 (m, 2H), 7.85 (t, J = 7.9 Hz, 1H), 7.69-7.58 (m, 5H), 7.20-7.14 (m, 1H), 7.05 (s, 1H), 6.95-6.91 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H). ESI-MS m/z 374.4 [M+H].
70		ESI-MS m/z 334.3 [M+H].
71		ESI-MS m/z 406.3 [M+H].
72		1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 8.31 (d, J = 4.0 Hz, 1H), 7.98 (d, J = 8.4Hz, 2H), 7.79-7.71 (m, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.52 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.01-6.95(m, 1H), 4.26 (s, 2H), 2.89(s, 3H), 1.70-1.36(m, 7H). ESI-MS m/z 351.5 [M+H].
73		1H NMR (400 MHz, DMSO) δ 11.56(s, 1H), 8.33 (d, J = 5.2Hz, 1H), 8.06 (d, J = 8.4Hz, 2H), 8.00 (t J = 8.0 Hz, 2H), 7.76 (t, J = 8.4 Hz, 1H), 7.68 (s, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.98 (t, J = 6.4 Hz, 1H), 2.60 (s, 3H). ESI-MS m/z 296.3 [M+H].
74		1H NMR (400 MHz, DMSO) δ 11.43 (s, 1H), 11.22 (s, 1H), 9.01 (s, 1H), 8.31 (d, J = 4.1 Hz, 1H), 8.04-7.95 (m, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.72 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.97-6.91 (m, 1H). ESI-MS m/z 313.3 [M+H].
75		1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.37 (d, J = 4.0 Hz, 1H), 7.97 (t, J = 17.5 Hz, 2H), 7.58-7.50 (m, 1H), 7.46 (dd, J = 10.1, 6.4 Hz, 3H), 7.25 (s, 2H), 7.10 (s, 1H), 6.88 (dd, J = 7.0, 5.2 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H). ESI-MS m/z 323.1 [M+H].
76		1H NMR (400 MHz, DMSO) δ 11.40 (s, 1H), 8.31 (d, J = 3.7 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.91 (dd, J = 2.7, 1.1 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.74 - 7.69 (m, 1H), 7.65 (dd, J = 5.0, 2.9 Hz, 1H), 7.61 (dd, J = 5.0, 1.0 Hz, 1H), 7.46 (s, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 6.9, 5.3 Hz, 1H). ESI-MS m/z 336.4 [M+H].
77		1H NMR (400 MHz, DMSO) δ 11.48 (s, 1H), 8.32 (d, J = 4.5 Hz, 1H), 8.09 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.83 - 7.58 (m, 2H), 7.11 (d, J = 8.3 Hz, 1H), 7.00-6.82 (m, 1H). ESI-MS m/z 322.3 [M+H].
78		1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.36 (d, J = 4.0 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.59 - 7.55 (m, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.07 (s, 1H), 6.89 (dd, J = 7.0, 5.2 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 3.12 (s, 1H). ESI-MS m/z 278.3 [M+H].
79		1H NMR (400 MHz, DMSO) δ 11.44 (s, 1H), 8.65 (d, J = 5.8 Hz, 2H), 8.33 (d, J = 4.7 Hz, 1H), 8.07 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 5.9 Hz, 2H), 7.73 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.99-6.91 (m, 1H). ESI-MS m/z 331.4 [M+H].
80		1H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.80 (s, 1H), 7.71 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.17 (d, J = 7.2 Hz, 2H), 7.00 (d, J = 6.0 Hz, 1H), 2.27 (s, 3H), 2.24 (s, 3H). ESI-MS m/z 282.3 [M+H].
81		1H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.80 (s, 2H), 7.71 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.26 (d, J = 8.0 Hz,1H),7.15 (d, J = 8.4 Hz,1H), 6.98 (t, J = 6.0 Hz,1H), 2.92-2.85 (m, 4H), 2.07-2.00 (m, 2H). ESI-MS m/z 294.3 [M+H].
82		1H NMR (400 MHz, DMSO) δ 11.67 (s, 1H), 8.36 (d, J = 4.9 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.37 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 6.0 Hz, 1H), 6.97 (d, J = 7.9 Hz, 1H), 6.05 (s, 2H). ESI-MS m/z 298.3 [M+H].
83		1H NMR (400 MHz, DMSO) δ 11.28 (s, 1H), 8.28 (d, J = 4.5 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.51 (s, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.90 (t, J = 5.6 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 4.97 (s, 2H), 2.10 (s, 3H). ESI-MS m/z 283.3 [M+H].

Test Example 1 UCP1 luciferase assay: Evaluating compounds that induce Ucp1 up-regulation.

[0063] Ucp1-luciferase knock-in mice will be housed in cages and fed on standard chow diets (the detailed procedures please see reference 14). The 8-week-old mice will be killed by the cervical dislocation and put into 75% alcohol for 5 minutes. The inguinal adipose tissue will be dissected out and transferred to the three 10-cm petri dishes and washed with PBS for three times. The adipose tissue will be dissected and minced on a 10-cm petri dish for several minutes and then pipetted into 20 ml 0.1% collagenase solution in 50ml conical tubes and digested at 37 °C for 1 hour. Next, 20 ml culture medium will be added to terminate the digestion and centrifuged for 10 minutes at 1000 rpm respectively. The supernatants will be removed and the pellets re-suspended in 10 mL culture medium and plated into 10-cm plates. The cells will be left to adhere to the plates overnight and the culture medium will be changed every other day. The confluent cells will be trypsinized, counted and plated to 24-well plate (5×10⁴cells/well). The cells will be washed and re-fed the next day (as day -2). Two days later (day 0), the cells will usually become confluent and be induced to differentiate with MDIR medium. On day 2, the medium will be changed to IR medium and re-fed every 2 days. Complete differentiation is usually achieved by day 10. On day 10, the medium will be changed to the culture medium of 10% FBS/DMEM with indicated drugs along with the positive and negative controls for 2 days. The media will be removed and the cells washed with 1 ml PBS per well three times. Then 100µl lysis buffer will be used to lyse the cells at 4°C for 1 hour. 30µl culture medium, 30µl cell lysates and 60µl Steady-Glo^® Reagent were added to a well of 96-well-plate and the luminescence will be measured using a luminometer. The protein concentration will be determined using 20µl cell lysates and specific activity of luciferase will be tabulated and data analyzed.

[0064] The bioactivity data of the compounds are listed in the following table.

Compound No.	Ucp1 activation fold/ corresponding concentration	Compound No.	Ucp1 activation fold/ corresponding concentration
1	5.2/3µM	43	2.8/1µM
2	1.1/1µM	44	1.9/3µM
3	2.8/1µM	45	1.3/0.3µM
4	4.1/3µM	46	1.7/3µM
5	5.4/3µM	47	2.4/3µM
6	1.2/1µM	48	6.0/3µM
7	1.2/0.3µM	49	3.9/1µM
8	4.0/1µM	50	2.4/3µM
9	3.6/1µM	51	3.9/1µM
10	3.8/3µM	52	5.1/1µM
11	2.0/1µM	53	3.0/1µM
12	1.2/1µM	54	3.9/1µM
13	1.6/3µM	55	2.0/3µM
14	2.4/3µM	56	2.3/3µM
15	1.4/0.3µM	57	3.5/3µM
16	5.1/3µM	58	1.4/3µM
17	1.9/1µM	59	4.0/1µM
18	5.3/3µM	60	2.6/3µM
19	1.6/1µM	61	3.2/3µM
20	3.0/3µM	62	2.2/3µM
21	2.9/3µM	63	5.9/3µM
22	3.9/1µM	64	1.1/1µM
23	4.0/3µM	65	1.1/3µM
24	2.7/3µM	66	1.0/3µM
25	1.3/3µM	67	3.1/3µM
26	3.5/3µM	68	1.3/3µM
27	3.3/1µM	69	4.6/3µM
28	3.2/3µM	70	1.7/0.3µM
29	1.2/0.3µM	71	1.7/0.3µM
30	1.1/1µM	72	1.8/3µM
31	1.1/1µM	73	1.2/3µM
32	2.6/3 µM	74	3.0/3µM
33	2.0/3µM	75	1.1/3µM
34	3.8/1µM	76	1.8/3µM
35	3.7/1µM	77	1.1/3µM
36	3.8/1µM	78	2.3/1µM
37	2.0/3µM	79	1.8/3µM
38	5.0/3µM	80	1.5/3µM
39	3.4/1µM	81	2.0/1µM
40	3.0/3µM	82	2.4/1µM
41	3.3/1µM	83	6.0/3µM
42	2.0/1µM

Test Example 2: high fat diet induced obesity mouse model

[0065] 8-week-old male mice (57BL/6J) were housed in metabolism cages and maintained on a 12 h light-dark cycle at 23°C and fed high fat diet (HFD) (21.9 kJ/g, 60% of energy as fat, 20% of energy as protein, 20% of energy as carbohydrate; D12492; Research Diet, New Brunswick, NJ, USA) for 8 weeks. Then, diets induced obese mice were randomized into two groups according to glucose levels and body weight and gavaged with daily indicated drugs (50 mg/kg) or vehicle. Food intake and body weight was measured weekly. All experiments with animals were approved by the Animal Care and Use Committee of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences.

Test Example 3: Hematoxylin and eosin staining

[0066] Hematoxylin and eosin staining was performed as the standard protocol (see reference 15). As to the results, please see figure 1 (left panel) using compound 1 as the test compound.

Test Example 4: Immunochemistry

[0067] Wash sections in dH₂O three times for 5 minutes each. Incubate sections in 3% hydrogen peroxide for 10 minutes. Wash sections in dH₂O twice for 5 minutes each, and then wash sections in wash buffer for 5 minutes. Block each section with 100-400 µl blocking solution for 1 hour at room temperature. Remove blocking solution and add 100-400 µl primary UCP1 antibody to each section. Incubate overnight at 4°C. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each. Add 100-400 µl biotinylated secondary antibodies, diluted in TBST to each section. Incubate 30 minutes at room temperature. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each. Add 100-400 µl DAB to each section and monitor staining closely. As soon as the sections develop, immerse slides in dH₂O. Wash sections in dH₂O two times for 5 minutes each. Incubate sections in 95% ethanol two times for 10 seconds each. Repeat in 100% ethanol, incubating sections two times for 10 seconds each. Repeat in xylene, incubating sections two times for 10 seconds each. Mount cover slips and take pictures. As to the results, please see figure 1 (right panel) using compound 1 as the test compound.

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9. Himms-Hagen, J., Melnyk, A., Zingaretti, M. C., Ceresi, E., Barbatelli, G. & Cinti, S. Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes. Am J Physiol Cell Physiol 279, C670-681, (2000).

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11. Vila-Bedmar, R., Lorenzo, M. & Fernandez-Veledo, S. Adenosine 5'-monophosphateactivated protein kinase-mammalian target of rapamycin cross talk regulates brown adipocyte differentiation. Endocrinology 151, 980-992, (2010).

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Claims

1. A compound or physiologically acceptable salt or hydrate or solvate thereof, wherein the compound is selected from the following compounds:

methyl 4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoate;

4-(4-amino-3-methylphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

3-(2-(pyridin-2-ylamino)thiazol-4-yl)benzonitrile;

N-(4-chloropyridin-2-yl)-4-(3,4-dimethoxyphenyl)thiazol-2-amine;

N-(pyridin-2-yl)-4-(4-(trifluoromethoxy)phenyl)thiazol-2-amine;

4-(4-bromo-2-methoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(2-bromo-4-morpholinophenyl)-N-(pyridin-2-yl)thiazol-2-amine;

1-isopropyl-3-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)urea;

N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)propionamide;

N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)butyramide;

4-(4-isopropoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-butoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

ethyl 2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetate;

2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetic acid;

1-morpholino-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)ethanone;

N-ethyl-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetamide;

4-(biphenyl-4-yl)-N-(pyridin-2-yl)thiazol-2-amine;

morpholino(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)methanone;

N-ethyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

4-(4-(morpholinomethyl)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-(benzylamino)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-(neopentylamino)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

N-isopropyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

N-phenyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

(4-methylpiperazin-1-yl)(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl) methanone;

4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzyl acetate;

1-(4-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)piperidin-1-yl)ethanone;

N-(2-(2-methoxyethoxy)ethyl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

4-(2-(pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-3-yl)benzamide;

4-(2-(pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-4-yl)benzamide;

N,N-diethyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

(S)-ethyl 1-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoyl)pyrrolidine-2-carboxylate;

N-(pyridin-2-yl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

4-(4-(piperidin-1-ylmethyl)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

1-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)ethanone;

N-hydroxy-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

N-(pyridin-2-yl)-4-(4-(thiophen-3-yl)phenyl)thiazol-2-amine;

4-(4-(2H-tetrazol-5-yl)phenyl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-ethynylphenyl)-N-(pyridin-2-yl)thiazol-2-amine;

N-(pyridin-2-yl)-4-(4-(pyridin-4-yl)phenyl)thiazol-2-amine.

2. A pharmaceutical composition comprising the compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1 and at least one pharmaceutically acceptable carrier or diluents.

3. The pharmaceutical composition of claim 2 wherein the composition is an oral composition, injectable composition or suppository.

4. The compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1 or the composition of claim 2 or 3 for use in treating metabolic disorders.

5. The compound or physiologically acceptable salt or hydrate or solvate thereof or the composition for use according to claim 4, wherein the metabolic disorder is obesity or diabetes.

Ansprüche

1. Verbindung oder physiologisch annehmbares Salz oder Hydrat oder Solvat hiervon, worin die Verbindung ausgewählt ist aus den folgenden:

Methyl 4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoat;

4-(4-Amino-3-methylphenyl)-N-(pyridin-2-yl)thiazol-2-amin;

3-(2-(Pyridin-2-ylamino)thiazol-4-yl)benzonitril;

N-(4-Chlorpyridin-2-yl)-4-(3,4-dimethoxyphenyl)thiazol-2-amin;

N-(Pyridin-2-yl)-4-(4-(trifluormethoxy)phenyl)thiazol-2-amin;

4-(4-Brom-2-methoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amin;

4-(2-Bromo-4-morpholinophenyl)-N-(pyridin-2-yl)thiazol-2-amin;

1-Isopropyl-3-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)harnstoff;

N-(4-(2-(Pyridin-2-ylamino)thiazol-4-yl)phenyl)propionamid;

N-(4-(2-(Pyridin-2-ylamino)thiazol-4-yl)phenyl)butyramid;

4-(4-Isopropoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amin;

4-(4-Butoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amin;

Ethyl 2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetat;

2-(4-(2-(Pyridin-2-ylamino)thiazol-4-yl)phenoxy)essigsäure;

1-Morpholino-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)ethanon;

N-Ethyl-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenoxy)acetamid;

4-(Biphenyl-4-yl)-N-(pyridin-2-yl)thiazol-2-amin;

Morpholino(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl)methanon;

N-Ethyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamid;

4-(4-(Morpholinomethyl)phenyl)-N-(pyridin-2-yl)thiazol-2-amin;

4-(4-(Benzylamino)phenyl)-N-(pyridin-2-yl)thiazol-2-amin;

4-(4-(Neopentylamino)phenyl)-N-(pyridin-2-yl)thiazol-2-amin;

N-Isopropyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamid;

N-Phenyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamid;

(4-Methylpiperazin-1-yl)(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phenyl) methanon;

4-(2-(Pyridin-2-ylamino)thiazol-4-yl)benzylacetat;

1-(4-(4-(2-(Pyridin-2-ylamino)thiazol-4-yl)phenyl)piperidin-1-yl)ethanon;

N-(2-(2-Methoxyethoxy)ethyl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamid;

4-(2-(Pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-3-yl)benzamid;

4-(2-(Pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-4-yl)benzamid;

N,N-Diethyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamid;

(S)-Ethyl-1-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoyl)pyrrolidin-2-carboxylat;

N-(Pyridin-2-yl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamid;

4-(4-(Piperidin-1-ylmethyl)phenyl)-N-(pyridin-2-yl)thiazol-2-amin;

1-(4-(2-(Pyridin-2-ylamino)thiazol-4-yl)phenyl)ethanon;

N-hydroxy-4-(2-(Pyridin-2-ylamino)thiazol-4-yl)benzamid;

N-(Pyridin-2-yl)-4-(4-(thiophen-3-yl)phenyl)thiazol-2-amin;

4-(4-(2H-Tetrazol-5-yl)phenyl)-N-(pyridin-2-yl)thiazol-2-amin;

4-(4-Ethynylphenyl)-N-(pyridin-2-yl)thiazol-2-amin;

N-(Pyridin-2-yl)-4-(4-(pyridin-4-yl)phenyl)thiazol-2-amin.

2. Pharmazeutische Zusammensetzung umfassend die Verbindung oder das physiologische Salz oder Hydrat oder Solvat hiervon gemäß Anspruch 1 und mindestens einen pharmazeutisch annehmbaren Träger oder Verdünnungsmittel.

3. Pharmazeutische Zusammensetzung gemäß Anspruch 2, worin die Zusammensetzung eine orale Zusammensetzung, injizierbare Zusammensetzung oder ein Suppositorium ist.

4. Verbindung oder physiologisch annehmbares Salz oder Hydrat oder Solvat hiervon gemäß Anspruch 1 oder Zusammensetzung gemäß Anspruch 2 oder 3 zur Verwendung bei der Behandlung von Stoffwechselerkrankungen.

5. Verbindung oder physiologisch annehmbares Salz oder Hydrat oder Solvat hiervon oder Zusammensetzung zur Verwendung gemäß Anspruch 4, worin die Stoffwechselerkrankung Fettsucht oder Diabetes ist.

Revendications

1. Composé ou sel physiologiquement acceptable ou hydrate ou solvate de celui-ci, où le composé est choisi parmi les composés suivants:

4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoate de méthyle;

4-(4-amino-3-méthylphényl)-N-(pyridin-2-yl)thiazol-2-amine;

3 -(2-(pyridin-2-ylamino)thiazol-4-yl)benzonitrile;

N-(4-chloropyridin-2-yl)-4-(3,4-diméthoxyphényl)thiazol-2-amine;

N-(pyridin-2-yl)-4-(4-(trifluorométhoxy)phényl)thiazol-2-amine;

4-(4-bromo-2-méthoxyphényl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(2-bromo-4-morpholinophényl)-N-(pyridin-2-yl)thiazol-2-amine;

100µl -isopropyl-3 -(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)urée;

N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)propionamide;

N-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)butyramide;

4-(4-isopropoxyphényl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-butoxyphényl)-N-(pyridin-2-yl)thiazol-2-amine;

2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phénoxy)acétate d'éthyle;

acide 2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phénoxy)acétique;

1-morpholino-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phénoxy)éthanone;

N-éthyl-2-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phénoxy)acétamide;

4-(biphényl-4-yl)-N-(pyridin-2-yl)thiazol-2-amine;

morpholino(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)méthanone;

N-éthyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

4-(4-(morpholinométhyl)phényl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-(benzylamino)phényl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-(néopentylamino)phényl)-N-(pyridin-2-yl)thiazol-2-amine;

N-isopropyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

N-phényl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

(4-méthylpipérazin-1-yl)(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)méthanone;

acétate de 4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzyle;

1-(4-(4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)pipéridin-100µl -yl)éthanone;

N-(2-(2-méthoxyéthoxy)éthyl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide; 4-(2-(pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-3-yl)benzamide;

4-(2-(pyridin-2-ylamino)thiazol-4-yl)-N-(pyridin-4-yl)benzamide;

N,N-diéthyl-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

(S)-100µl -(4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzoyl)pyrrolidine-2-carboxylate d'éthyle;

N-(pyridin-2-yl)-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

4-(4-(pipéridin-1-ylméthyl)phényl)-N-(pyridin-2-yl)thiazol-2-amine;

1-( 4-(2-(pyridin-2-ylamino)thiazol-4-yl)phényl)éthanone;

N-hydroxy-4-(2-(pyridin-2-ylamino)thiazol-4-yl)benzamide;

N-(pyridin-2-yl)-4-(4-(thiophén-3-yl)phényl)thiazol-2-amine;

4-(4-(2H-tétrazol-5-yl)phényl)-N-(pyridin-2-yl)thiazol-2-amine;

4-(4-éthynylphényl)-N-(pyridin-2-yl)thiazol-2-amine;

N-(pyridin-2-yl)-4-(4-(pyridin-4-yl)phényl)thiazol-2-amine.

2. Composition pharmaceutique comprenant le composé ou sel physiologiquement acceptable ou hydrate ou solvate de celui-ci selon la revendication 1 et au moins un vecteur ou diluant pharmaceutiquement acceptable.

3. Composition pharmaceutique selon la revendication 2, où la composition est une composition orale, une composition injectable ou un suppositoire.

4. Composé ou sel physiologiquement acceptable ou hydrate ou solvate de celui-ci selon la revendication 1 ou composition selon la revendication 2 ou 3 destiné à être utilisé dans le traitement de troubles métaboliques.

5. Composé ou sel physiologiquement acceptable ou hydrate ou solvate de celui-ci ou composition destiné à être utilisé selon la revendication 4, où le trouble métabolique est l'obésité ou le diabète.

Drawing